Figure 6.

Freeze fracture electron micrograph analysis of HEK293 cells stably expressing claudin-10 constructs. (A) Claudin-10a WT forms meshworks of continuous tight junction (TJ) strands (white arrows) detected on the protoplasmic fracture face (PF) of the plasma membrane. On the exoplasmic fracture face (EF), complementary continuous grooves are evident (white arrowheads). (B) For claudin-10a G163A, similar continuous TJ strands (white arrows) on the PF and grooves (white arrowheads) on the EF are detected to some extent. (C) In most instances, however, discontinuous claudin-10a G163A TJ strands (black arrows) are detected in the form of beaded particles on the PF. The abundance of strands appears to be lower than for claudin-10a WT. (D, E) As is the case with claudin-10a WT, claudin-10b WT forms meshworks of continuous TJ strands (white arrows) on the PF and complementary continuous grooves on the EF (white arrowheads). (F) In contrast, for claudin-10b G165A, fewer and highly discontinuous, fragmented TJ strands (black arrows) can be detected as beaded particles on the PF. On the EF, irregular grooves, pits, and particles are evident (black arrowheads). (G) For claudin-10b G165A, only in few cases, short continuous TJ strands (white arrows) are observed on the PF. Representative images; scale bars, 200 nm. (H) Quantification of strand breaks. Percentages of strand breaks are higher in the mutant cases. In addition, claudin-10b G165A strands exhibit higher break percentages than do claudin-10a G163A strands. Along strands, the particle-free distances between particles or strand segments were determined. The percentage of strand breaks was calculated from the quotient total particle-free length/total strand length. For each sample, n = 10 electron micrographs were analyzed. Kruskal-Wallis test followed by post-hoc two-tailed Mann Whitney tests with Holm-Šídák multiple comparisons test; ∗, P < 0.05; ∗∗∗, P < 0.001. Mean ± SEM. WT, wild-type.